Abstract

Background There is no effective medical treatment for heart failure with preserved ejection fraction (HFpEF). Increases in pulmonary capillary wedge pressure (PCWP) develop in patients with HFpEF during exercise coupled with impaired nitric oxide (NO) signaling. Nitrite can be reduced to bioactive NO in vivo, particularly under conditions of tissue hypoxia, as with exercise.

Approximately one-half of patients with heart failure have heart failure with preserved ejection fraction (HFpEF), and there is no effective treatment (1). During exercise, the normal left ventricle can fill to a larger diastolic volume with no increase in pressure, but in individuals with HFpEF, a marked increase in left ventricular (LV) filling pressures with exercise develops, contributing to increases in morbidity and mortality (2–9). Many patients with HFpEF also display impaired cardiac output (CO) reserve with exercise, which further contributes to exercise intolerance (6–8,10,11). Thus, increased filling pressures and inadequate CO reserve represent viable targets for new interventions designed to improve symptoms and outcome in HFpEF.

Numerous lines of evidence indicate that abnormalities in nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) signaling play a central role in causing these reserve limitations (11–13). Organic nitrates can improve NO-cGMP signaling but may be limited by the development of tolerance or symptomatic hypotension (14,15). Indeed, 1 factor complicating HFpEF treatment is that the hemodynamic perturbations causing symptoms are often absent at rest but observed only during physiological stresses, such as exercise (2).

Inorganic nitrite is now recognized as an alternative in vivo source of NO-cGMP that is independent of the traditional NO synthase pathway (16–19). Intriguingly, the reduction of nitrite to bioactive NO may be enhanced by tissue hypoxia and acidosis (19), which develop during exercise. This suggests that nitrite might more selectively target hemodynamic derangements developing during stress in people with HFpEF (2,10), with less risk of hypotension at rest (15). The current study tests the hypothesis that, compared with placebo, acute infusion of sodium nitrite would improve exercise hemodynamics and enhance cardiac reserve in patients with HFpEF.

Methods

This double-blind, randomized, placebo-controlled, parallel-group trial was designed to study the effects of intravenous sodium nitrite on cardiovascular hemodynamics at rest and during exercise in subjects with HFpEF. Patients referred to the Mayo Clinic cardiac catheterization laboratory for invasive hemodynamic exercise stress testing were enrolled. Written informed consent was provided by all subjects before participation in study-related procedures. The Mayo Clinic Institutional Review Board approved the study.

Subjects were studied on their long-term medications in the post-absorptive state and supine position. Cardiac catheterization was performed with simultaneous expired gas analysis at rest and during supine exercise at a 20-W workload for 5 min, as previously described (2,8). After the first exercise phase (before any drug administration) and after return to steady-state baseline hemodynamic values, subjects were randomized 1:1 to infusion of placebo (normal saline solution) or sodium nitrite (50 μg/kg/min) (Hope Pharmaceuticals, Scottsdale, Arizona) for 5 min. The nitrite/placebo infusions were identical in appearance and prepared by the research pharmacy, ensuring double-blinding of infusion content. After 10 min, hemodynamic measurements were repeated at rest, followed by repeat supine exercise at a 20-W workload for 5 min, identical to the study’s first phase. Arterial and venous blood samples and hemodynamic and expired gas data were acquired during each stage of the protocol.

Right heart catheterization was performed through a 9-F sheath via the internal jugular vein. Transducers were zeroed at mid-axilla. Right atrial pressure (RAP), pulmonary artery (PA) pressure, and PCWP were measured at end-expiration (mean of ≥3 beats) using 2-F, high-fidelity micromanometer-tipped catheters (Millar Instruments, Houston, Texas) advanced through the lumen of a 7-F, fluid-filled catheter (Arrow, Teleflex, Morrisville, North Carolina) (2,8). Mean RAP and PCWP were taken at mid A wave. PCWP position was verified by typical waveforms, appearance on fluoroscopy, and direct oximetry (saturation ≥94%). Continuously recorded pressure tracings were digitized (240 Hz) and analyzed offline.

Central venous and arterial blood samples were obtained during each stage to measure the methemoglobin level and blood gases. Plasma nitrite concentrations were assessed in the final 6 subjects enrolled in the trial using a liquid chromatography–fluorometric assay (BASi, West Lafayette, Indiana) as previously described (20).

Study endpoints

The primary endpoint of the trial was the PCWP during exercise. Secondary endpoints included changes in resting PCWP as well as rest and exercise changes in RAP, PA pressure, PVR, PA compliance, systemic BP, heart rate, SV, stroke work, CO, Vo2, and Cao2 − Cvo2. Adequacy of CO reserve was assessed by comparing the CO/Vo2 slope before and after study drug infusion, whereas PA pressure-flow relationships were assessed to integrate changes in right heart loading with exercise. Methemoglobin level (%) was assessed as a safety endpoint.

Statistical analysis

Results are reported as mean ± SD, median (interquartile range) or n (%). Between-group differences at individual time points were tested using the Student t test, Wilcoxon rank sum test, or Fisher exact test. Within-group differences are assessed by the paired Student t test. The effect of nitrite on the primary endpoint of exercise PCWP was assessed by analysis of covariance, using the initial exercise PCWP measured before study drug infusion as the covariate. Between-group differences in rest or exercise hemodynamic responses were compared by an unpaired Student t test after accounting for respective pre-study drug values. Linear regression was performed to compare hemodynamic responses to exercise before and after study drug infusion, with variables log-transformed as necessary for analysis. All tests were 2-sided, with p < 0.05 considered significant. Analyses were performed using JMP version 10.0.0 (SAS Institute, Cary, North Carolina).

Results

A total of 28 subjects were enrolled in the trial between January and September 2014. Baseline characteristics were not significantly different between treatment groups (Table 1). Subjects were older, obese, and predominantly female, with a high prevalence of hypertension. On average, subjects displayed normal LV chamber size and mass, left atrial enlargement, mild LV diastolic dysfunction, and increased N-terminal pro–B-type natriuretic peptide levels (Table 1).

The increase in CO relative to metabolic demand (ΔCO/ΔVo2 slope) in the HFpEF subjects was 4.7 ± 2.7 ml/ml. Most participants (75%) displayed an abnormal ΔCO/ΔVo2 slope (defined as <6 ml/ml) (21), indicating significant limitation in CO reserve during exercise. The median slope of increase in PA pressure relative to CO (ΔPA/ΔCO slope) was 9.4 (interquartile range: 5.3 to 14.3) mm Hg/l/min. The vast majority of subjects (93%) displayed abnormal PA pressure-flow relationships with exercise (defined as >3.0 mm Hg/l/min) (22). There were no between-group differences in any of the indexes of resting or exercise hemodynamics, expired gas data, or ventricular function.

Nitrite effects

Hypotension or other adverse events after study drug infusion did not develop in any subjects. Nitrite modestly increased methemoglobin levels compared with placebo (+0.5 ± 0.3% vs. +0.1 ± 0.4%; p = 0.002), but no clinically meaningful methemoglobinemia developed in any subject (>5%). The highest methemoglobin level observed (2.4%) was in a subject randomized to nitrite with an increased resting level (1.6%).

The increase in exercise CO with nitrite was caused exclusively by greater enhancement in SV (Figure 3C), as there was no effect on exercise heart rate (Table 4). Although the enhanced SV reserve with nitrite might have been related in part to systemic vasodilation (lower SVR), importantly, there was also a greater increase in LV stroke work with nitrite (Figure 3D), indicating an acute increase in LV systolic performance, independent of changes in cardiac loading (23).

Plasma nitrite levels

Venous plasma nitrite levels were obtained in 4 subjects randomized to nitrite and 2 randomized to placebo. Levels were undetectable at baseline and with exercise before study drug infusion in all subjects. Nitrite levels remained undetectable after study drug infusion in subjects receiving placebo, but increased to 8.39 ± 1.88 μM at rest in subjects receiving active drug (p = 0.004 vs. baseline). After the 5-min exercise period, levels decreased to 3.36 ± 0.42 μM in the active therapy group (p < 0.01 compared with pre-exercise values). The calculated half-life from these data was 3.9 ± 0.6 min, which is 10-fold faster than the previously reported kinetics for sodium nitrite in humans at rest (30 to 40 min) (20), indicating active consumption during exercise.

Discussion

This double-blind, randomized, placebo-controlled trial tested the effects of acute infusion of inorganic sodium nitrite on cardiovascular hemodynamics and ventricular function at rest and during low-level exercise in subjects with HFpEF. The rationale for this design was based on the fact that hemodynamic derangements in patients with HFpEF often develop only during exercise, when a reduction of nitrite to NO is believed to be enhanced because of tissue hypoxia. The primary endpoint of exercise PCWP was significantly improved by nitrite, resulting in a 37% reduction in left heart filling pressures with exercise (Central Illustration). Beneficial reductions in PCWP were coupled to improvements in exercise CO reserve, reductions in PA pressures and PA pressure-flow relationships, and enhanced systemic vasodilator reserve.

Importantly, nitrite therapy was associated with beneficial myocardial effects in addition to vascular effects, evidenced by a greater increase in LV stroke work with exercise, an integrated index of LV diastolic and systolic performance. Beneficial effects on hemodynamics and ventricular function were of greater magnitude during exercise compared with rest, and, among participants with nitrite levels assessed, there was greater than expected decay in nitrite levels during exercise, consistent with active nitrite consumption. The beneficial effects of acute nitrite infusion on multiple hemodynamic derangements developing during exercise in HFpEF provides compelling rationale to pursue longer term clinical trials of inorganic nitrites in patients with HFpEF, a population for whom there is currently no effective treatment.

Pathophysiology of HFpEF and rationale for no-enhancing therapies

The pathophysiology of HFpEF is complex, related to abnormalities in LV diastolic function and diastolic reserve as well as to limitations in systolic reserve, abnormal peripheral and pulmonary vasodilation, endothelial dysfunction, chronotropic incompetence, right ventricular dysfunction, and, as recently shown, limitations in the periphery (1–8,10,11,24–26). The subjects enrolled in the current study displayed many of these hemodynamic abnormalities, with a slight increase in resting RAP, PA pressure, and PCWP and dramatic increases during exercise that were coupled to limitations in CO reserve and abnormal pulmonary vascular function. These hemodynamic abnormalities importantly contribute to central congestion and inadequate tissue perfusion during stress and, thus, represent viable targets for therapeutic intervention.

Previous trials testing inhibitors of the renin-angiotensin-aldosterone system failed to show benefit in HFpEF, and there is currently no proven treatment (1). Numerous lines of evidence point to limitations in NO-cGMP as playing a key role in determining the functional and hemodynamic abnormalities developing in HFpEF (11–13). Sildenafil, an inhibitor of phosphodiesterase-5 (which catabolizes cGMP), did not enhance exercise capacity or clinical status in a recent multicenter trial (27). However, elegant work from van Heerebeek et al. (12) showed that cGMP limitation in HFpEF is not related to excessive breakdown but rather inadequate production, suggesting that NO-cGMP–providing therapies may be the more effective approach. A major barrier in managing many patients with HFpEF is related to the fact that the increase in cardiac filling pressures and PA pressure is confined to exercise, whereas hemodynamics at rest may be more normal (1–8). Thus, an agent that enhances NO-cGMP signaling preferentially during exercise would be expected to provide more targeted hemodynamic improvements precisely at the time of greatest need.

The nitrate-nitrite-no pathway in heart failure

Inorganic nitrite and nitrate were previously considered to be inert byproducts of NO metabolism, but recent work has shown these serve as an important in vivo reservoir of NO (16–19). Dietary nitrate is absorbed, secreted in saliva, and then converted by oral bacteria to nitrite, which is absorbed and then reduced by a number of enzymes, including deoxygenated hemoglobin or myoglobin, to NO (19,28,29). This reaction is believed to be enhanced in the setting of hypoxia and acidosis, which develop in the tissues and venous circulation during exercise, potentially allowing for hypoxic vasodilation that complements the alternative oxygen-dependent NO synthase pathway (16,18,19). In contrast to the organic nitrates that require aldehyde dehydrogenase and other enzymes for activation (14), there is no tolerance with nitrate-nitrite (17). The greater reduction in SVR with exercise in the current study is consistent with previously described vasodilatory effects in humans, and the fact that SVR reduction was only observed during exercise is consistent with the hypoxia potentiation of nitrite effect (16–18).

Recent studies have begun to explore the potential role for the nitrate-nitrite-NO pathway in the treatment of heart failure. Zamani et al. (30) performed a noninvasive double-blind, crossover study in 17 subjects with HFpEF comparing nitrate-rich beetroot juice with nitrate-depleted beetroot juice. The authors observed significant improvements in peak Vo2, peak exercise workload, exercise CO (assessed by echocardiography), and exercise vasodilation (reduction in SVR) (30). The improvement in CO reserve observed with nitrate therapy was due to a greater increase in heart rate, with no significant effect on SV.

The current invasive hemodynamic data extend the noninvasive findings of Zamani et al. (30), showing greater increases in CO and improved systemic vasodilation with nitrite in HFpEF subjects, assessed using gold-standard techniques. The increase in CO noted in the current study greatly exceeded the small increase in Vo2, indicating an acute increase in the ability of the heart to provide blood flow relative to metabolic needs (CO/Vo2 slope), which has been shown to be impaired on average in HFpEF (10). In contrast to Zamani et al. (30), the enhanced CO reserve observed in the current study was related to a greater increase in SV with exercise, with no effect on rest or exercise heart rate.

Although the greater increase in SV in the current study was likely mediated in part by improved afterload reduction, we also observed beneficial effects of nitrite on ventricular performance, assessed by the greater increase in LV stroke work with exercise. Stroke work is independent of afterload, but varies directly with preload (end-diastolic volume) (23). Therefore, we cannot determine whether the beneficial effect of nitrite on LV performance in the current study was mediated by improvements in diastolic reserve (greater increase in end-diastolic volume despite lower filling pressures), systolic reserve (increased contractility), or both. However, given the fact that both diastolic and systolic reserves are known to contribute to the pathophysiology of HFpEF, the observation of a direct myocardial benefit from nitrite is an important observation.

The main novel finding and primary endpoint of the trial was the greater reduction in PCWP during exercise, a key force mediating symptoms of exertional dyspnea in people with HFpEF. The magnitude of decrease in exercise PCWP with nitrite was much greater than the resting PCWP, consistent with greater effectiveness of nitrite during exercise, as noted earlier. This represents an important advantage of inorganic nitrite for HFpEF patients, especially those with early-stage disease in which PCWP is normal at rest and increased only during exercise. The decrease in exercise PCWP was coupled with significant reductions in RAP and PA pressure with stress, and an acute decrease in the slope of the PA pressure-flow relationship, indicating an improvement in right ventricular afterload (22). Given the enhanced afterload sensitivity of the right ventricle in HFpEF, this decrease in PA pressure would be expected to greatly improve right ventricular performance as well (24).

Although decreases in rest and exercise PA pressure were observed, there was no significant effect of nitrite on PVR or PA compliance at rest or during exercise. Several animal studies have observed improvements in PA pressure and vascular remodeling with nitrate or nitrite, but most of these model systems are characterized by more severe pulmonary vascular disease (31). Subjects in the current study displayed pulmonary vasoconstriction compared with normal reference values (32), but increases in PVR and decreases in PA compliance were relatively modest and not in the range associated with adverse outcomes in heart failure patients (33). Mean PA pressure-flow slopes were extremely increased in the study subjects, but this was predominantly due to post-capillary (pulmonary venous) disease. Mean PA pressure is equal to the product of CO and PVR summed with downstream PCWP. In patients with advanced pulmonary vascular disease, PA pressure is high because of increased PVR, but in the current study, increased PA pressure-flow slopes were caused almost exclusively by high PCWP. The current data may not be applicable to patients with pulmonary hypertension.

Other groups have noted improvements in the O2 cost of exercise with inorganic nitrite (34,35) in contrast to the current study in which Vo2 during submaximal exercise was slightly enhanced. The reason for the discrepancy is not clear, but may relate to the level of work performed and the subjects studied. For example, Larsen et al. (35) studied young healthy, well-trained men who were exercising at 4-fold higher Vo2 (3 l/min) than in the current study’s elderly, predominantly female HFpEF population (0.7 l/min). The current results showing an increase in Vo2 with nitrite during submaximal exercise in HFpEF agree with maximal exercise data from Zamani et al. (30). We speculate that greater increases in CO reserve from nitrite, possibly coupled with nitrite-mediated improvements in skeletal muscle microperfusion (36), are associated with less anaerobic glycolysis in exercising muscles in HFpEF patients and that this effect outweighs any ostensible lowering of the O2 cost of exercise. This question merits further study.

Plasma nitrite levels were assessed in a minority of subjects, including 4 randomized to nitrite and 2 to placebo. Levels were below assay at rest in all subjects and after study drug infusion in those receiving placebo. This is consistent with the low level of plasma nitrite levels in humans under normal circumstances (0.05 to 0.3 μM) (19). In subjects receiving active drug, a marked increase in plasma nitrite to >8 μM developed, approximately 2-fold higher than what has been observed with oral formulations (37). Although the low number of subjects with nitrite levels measured limits our ability to examine pharmacokinetics, it is notable that the observed elimination half-life during 5 min of exercise was 10-fold faster than what has been reported in resting humans (20). This is consistent with greater-than-normal consumption of nitrite during exercise in the study participants, presumably via greater reduction to NO with stress.

Clinical implications

Inorganic nitrate-nitrite could be applied therapeutically through naturally occurring sources such as beetroot juice or using oral supplements, although these routes would not achieve plasma nitrite levels in the range of those observed in the current study. Nitrite can also be administered orally and via an inhaled, nebulized device. Either of these routes may be suitable for chronic administration in people with HFpEF. Because of the rapid onset of action, the nebulized preparation may prove useful as a rescue inhaler or taken prophylactically before planned physical activity to reduce symptoms of exercise intolerance. The acute effects of inhaled nitrite on hemodynamics are currently being tested in HFpEF (NCT02262078), and larger phase II studies in this cohort are in the planning stages.

Organic nitrates such as isosorbide mononitrate also can enhance NO-cGMP and are commonly used in managing people with HFpEF (38). However, organic nitrates are limited by the development of tolerance, increases in oxidative stress, and the development of endothelial dysfunction (14). Organic nitrate tolerance can be reduced by ensuring an adequate nitrate-free window, but renal sodium retention may offset the beneficial effects of long-term venodilation. In contrast, there is no tolerance with inorganic nitrite, and, because conversion of nitrite to NO occurs preferentially during exercise, there may be less sodium retention and less risk of excessive BP reduction at rest, which can be problematic in those with HFpEF (15). The effect of organic nitrates on activity tolerance and exercise capacity is currently being investigated in the NEAT-HFpEF (Nitrate's Effect on Activity Tolerance in Heart Failure With Preserved Ejection Fraction) trial (38).

Study limitations

This study examined hemodynamics and LV performance at rest and with low-level exercise but not at peak exercise. This was done primarily for feasibility, as it would be difficult for subjects to complete 2 maximal-effort exercise tests in a timely fashion. Thus, we cannot determine whether benefits observed during low-level exercise would extend to peak exercise, although the greater venous hypoxia and acidosis at peak would only be expected to further potentiate the benefit. Furthermore, this level of activity is clinically meaningful in that it reflects the level of physical work performed in activities of daily life in typical older people with HFpEF. Right ventricular function and right ventricle–PA coupling may be improved by nitrite, but they were not assessed in this study.

Conclusions

Inorganic nitrite favorably attenuates hemodynamic derangements that develop during exercise in individuals with HFpEF, including increased cardiac filling pressures, exercise-induced pulmonary hypertension, and inadequate CO reserve. Beneficial effects of nitrite are mediated by both vascular unloading and direct myocardial effects, which are more pronounced during exercise compared with steady state. Prospective trials testing chronic nitrite therapy in patients with HFpEF are warranted.

Perspectives

COMPETENCY IN MEDICAL KNOWLEDGE: Sodium nitrite, a source of NO, improves hemodynamics during exercise in patients who have HFpEF, supporting a pathophysiological role for abnormal NO signaling in this condition.

TRANSLATIONAL OUTLOOK: Randomized trials of nitrite therapy are needed to evaluate longer term effects on functional capacity and clinical outcomes in patients with HFpEF.

Footnotes

This study was supported by a grant from the Mayo Clinic Division of Cardiovascular Diseases. Dr. Borlaug receives research support from Aires Pharmaceuticals for a separate study examining the use of inhaled nitrite in patients with HFpEF. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

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